The present invention relates to methods for efficient and reliable construction of adenovirus vectors that contain and express foreign DNA and are useful for gene transfer into mammalian cells, for vaccines and for gene therapy. The vector system described herein is an improvement and modification of the two plasmid pBHG system, described in copending patent application Ser. No. 08/250,885, a foreign equivalent of which published as WO95/00655, hereby incorporated by reference, whereby a replication defective genomic adenoviral vector and a shuttle plasmid were recombined via homologous recombination in a cell in which they were cotransfected. This invention further represents an improvement over the Ser. No. 09/263,650 system whereby, through insertion of a head-to-head ITR junction in the shuttle plasmid, enhanced vector rescue efficiency is achieved, whether through homologous or site-specific recombination mechanisms.
As taught in WO95/00655, adenoviruses (Ads) can be used as mammalian cell expression vectors, with excellent potential as live recombinant viral vaccines, as transducing vectors for gene therapy, for research, and for production of proteins in mammalian cells.
In the human Ad genome, early region 1 (E1), E3, and a site upstream of E4 have been utilized as sites for introducing foreign DNA sequences to generate adenovirus recombinants. In the absence of compensating deletions in E1 or E3, a maximum of about 2 kb can be inserted into the Ad genome to generate viable virus progeny. The E1 region is not required for viral replication in complementing 293 cells, or other cells known to complement E1, and up to approximately 3.2 kb can be deleted in this region to generate conditional helper independent vectors with a capacity of 5.0-5.2 kb. In the E3 region, which is not required for viral replication in cultured cells, deletions of various sizes have been utilized to generate nonconditional helper independent vectors with a capacity of up to 4.5-4.7 kb. The combination of deletions in E1 and E3 permits the construction and propagation of adenovirus vectors with a capacity for insertions of up to approximately 8 kb of foreign DNA.
The construction of Adenovirus vectors can be performed in many ways. One approach is to cotransfect permissive cells, usually, but not limited to, 293 cells, with a shuttle plasmid containing a portion of the left end of the Ad genome and, most commonly, having the E1 sequences replaced by a foreign DNA, and with DNA isolated from virions cleaved near the left end by a suitable restriction enzyme. Homologous recombination between overlapping viral DNA sequences of the shuttle plasmid and the virion DNA results in production of recombinant viruses containing the foreign DNA. A disadvantage of this method is the need to prepare purified viral DNA. In addition, such methods typically result in the presence of contaminating parental virus in the resulting vector preparations, such as when 100% of the viral DNA is not cleaved, or when the two viral DNA fragments produced by restriction cleavage are rejoined.
Another method has recently been described (Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps M L, xe2x80x9cConstruction of adenovirus vectors through Cre-lox recombination.xe2x80x9d J Virol March 1997; 71(3):1842-1849; see also PCT publication WO97/32481 relating to use of site-specific recombination of virus and helper dependent vectors) which involves infection of 293Cre cells (293 cells engineered to express Cre recombinase) with an Adenovirus containing a floxed packaging signal (xcexa8) and transfection with a shuttle plasmid containing an ITR, a packaging signal and an expression cassette followed by a lox site, or cotransfection of 293Cre cells with purified deproteinized Adenoviral DNA and a shuttle plasmid. According to that method, Cre-mediated excision of the packaging signal from virus followed by site-specific recombination with the lox site in the shuttle plasmid produces a recombinant vector containing the expression cassette. However, as Cre action is not 100% efficient, the resulting virus preparations remain contaminated with parental virus, and must be passaged in 293Cre cells to eliminate the contaminating parental virus. A further disadvantage of this method is that it requires use of an infectious virus or DNA extracted from a virus as one of the starting materials, and is thus less attractive for commercial distribution than kits containing only bacterial plasmid DNA. Furthermore, the parental virus can recombine with Ad E1 sequences present in 293 cells, resulting in a virus containing a wild-type packaging signal and a wild-type E1 region. Such recombinant virus has the propensity to overgrow the original vector, leading to contamination of subsequent vector preparations with non-attenuated E1 expressing Ads.
One of the most frequently used and most popular methods for construction of adenovirus vectors is based on xe2x80x9cthe two plasmid methodxe2x80x9d (see Bett, A. J., Haddara, W., Prevec, L. and Graham, F. L An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc. Natl. Acad. Sci. US 91: 8802-8806, 1994), whereby suitable host cells (typically 293 cells) are cotransfected with two plasmids that separately are incapable of generating infectious virus, but which, when recombined within the transfected cell by homologous recombination, can generate replicating virus. The most widely used plasmids of this type are described in patent application Ser. No.08/250,885, and in PCT publication number WO95/00655, hereby incorporated by reference. That system has advantages over other methods using viruses or viral DNA as components since only easily-prepared plasmid DNAs are needed, and there is no background of parental virus that could contaminate the final vector isolates. Furthermore, the plasmids are not only easy and inexpensive to produce by those skilled in the art, but can be easily stored and transported, making them convenient for commercial distribution, (i.e. particularly when precipitated with ethanol or when lyophilized, these vectors do not require a cold chain for distribution). However, although this currently available system has proven utility and is widely used, the efficiency of virus production by homologous recombination can be low and variable, and the system cannot always be used easily by those not skilled in the art.
As demonstrated in the art (Anton, M. and Graham, F. L. xe2x80x9cSite-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression,xe2x80x9d J. Virol. 69:4600-4606, 1995), and as described also in application Ser. No. 08/486,549 (xe2x80x9cAdenoviruses for control of gene expressionxe2x80x9d, hereby incorporated by reference), provision of Cre recombinase in Ad-infected cells can catalyse excision or rearrangement of viral DNA sequences that contain the target sites (lox P) for Cre-mediated site-specific recombination.
Such techniques are combined in the present invention with a head-to-head ITR containing shuttle plasmid to provide a long-needed advancement in the art of adenoviral vector production by enhancing the efficiency of vector production, whether through homologous recombination or site-specific recombination events.
In the present invention, viruses, plasmids or both are constructed which contain head-to-head ITR junctions, and optionally, wherein said viral DNA may also contain lox P sites positioned such that site-specific recombination between lox P sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Such cells (293Cre cells) have been described by Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. and Graham, F. L. xe2x80x9cA new helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal,xe2x80x9d Proc. Natl. Acad. Sci. U.S. 93: 13565-13570, 1996, by Chen, L., Anton, M. and Graham, F. L., xe2x80x9d Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre,xe2x80x9d Somat. Cell and Molec. Genet. 22: 477-488, 1996, in U.S. patent application Ser. No. 08/473,168, and in PCT publication WO96/40955, hereby incorporated by reference for this purpose. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing a contaminating parental adenovirus, with the attendant problems for removal thereof.
Due to the enhancement of recombination efficiency achieved by the instant invention, whereby a head-to-head ITR junction is included in the shuttle plasmid, even in the absence of site-specific recombination, enhanced efficiency is achieved in production of recombinant virus vectors.
In one embodiment of this invention a head-to-head ITR junction is included in the shuttle plasmid, whereby enhanced efficiency of production of recombinant virus vectors is achieved whether by homologous recombination or by site-specific recombination.
In a further embodiment of this invention, homologous recombination via cellular enzymes is replaced by site-specific recombination, via a recombinase such as Cre, FLP or the like, to join two DNAs that separately are noninfectious to form an infectious DNA molecule, wherein the efficiency of the site-specific recombination is enhanced by inclusion of head-to-head ITR junctions in the shuttle plasmids of a two-plasmid system. One application of the techniques disclosed herein is the isolation of xe2x80x9cfirst generationxe2x80x9d vectors with insertions of foreign DNA in E1. Such applications utilize a series of plasmids such as pBHGloxxcex94E1,3 (see FIG. 1, and variations and equivalents thereof), and various shuttle plasmids containing a head-to-head ITR junction, a packaging signal, an expression cassette, and a lox or other recombinase recognition site. Another application is in a sense the mirror image. Using a plasmid such as pFG173lox, sequences are rescued into the right end of the viral DNA, into E3 or into sites rightward of E3. The most important applications of this latter technology will likely be rescue of mutations into the fiber gene located immediately rightward of E3 (FIG. 9A) (fiber is important because it is a major ligand for binding to cellular receptors) but one can also rescue mutations, deletions, insertions and other modifications in E4 genes (located between fiber and the right ITR) or the method is used to rescue inserts of foreign DNA into E3 (cotransfection of a plasmid such as that depicted in FIG. 11 with pFG173lox). Note that the plasmid pFG173lox has a deletion of fiber, but E4 sequences could just as well be deleted as well as or instead of fiber. Note also that lox sites could be inserted at other locations in the Ad genome to enable the rescue of mutations engineered in other viral genes besides those of fiber or E4, or rescue of DNA inserts into other sites.
In a further embodiment of this invention, DNA-TP complexes are utilized to combine the high efficiency of head-to-head ITR mediated recombination, with or without site-specific recombination, such as Cre-lox recombination, with the high infectivity of DNA-TP. While the rescue of infectious virus via head-to-head ITR mediated homologous recombination, or head-to-head ITR mediated recombination with Cre-mediated recombination is surprisingly efficient compared to homologous recombination alone, and is more than adequate to produce viral vectors and to introduce mutations into the viral genome for most applications, there may be certain applications for which even higher efficiencies are desirable or necessary. It is known by those skilled in the art that the infectivity of adenovirus DNA is up to 100 fold higher if the virion DNA is extracted and purified by methods that leave intact the terminal protein (TP) that is normally linked to the 5xe2x80x2 end of each strand of the duplex Ad DNA molecule (Sharp P A, Moore C, Haverty J L, xe2x80x9cThe infectivity of adenovirus 5 DNA-protein complex,xe2x80x9d Virology December 1976;75(2):442-456, Chinnadurai G, Chinnadurai S, Green M, xe2x80x9cEnhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex.xe2x80x9d J Virol April 1978; 26(1):195-199). For rescue of cassettes, the two plasmid system is more than sufficiently efficient, especially with the approximately 10-fold enhancement in efficiency demonstrated herein for head-to-head ITR mediated recombination, or the approximately 100-fold enhancement in efficiency demonstrated herein for head-to-head ITR mediated recombination when coupled with Cre-lox mediated recombination (over homologous recombination alone), and consequently would be preferred for most purposes. However, there may be times when even higher efficiencies are required, as when, for example, one wishes to develop a library of fiber mutations (a large number of different virusesxe2x80x94the more the better). Then the chore of preparing DNA-TP might be worthwhile and could be accomplished by those skilled in the art. Thus, an aspect of the present invention includes the combination of the enhanced recombination mediated by head-to-head ITR junctions in shuttle plasmids, with or without Cre-lox recombination, with the high specific infectivity of adenoviral DNA-TP complexes.
Therefore, it is an object of the present invention to provide a highly efficient, reliable, and simple method for isolation of viral vectors in which rescue of viruses is enhanced by provision of head-to-head ITR junctions whether recombination is based on homologous recombination, or on site-specific recombination catalysed by a site-specific recombinase, such as but not limited to the Cre recombinase.
It is a further object of this invention to use head-to-head ITR constructs, with or without Cre-lox-mediated recombination, and known two plasmid vector production systems to provide a simple method for introducing mutations or other modifications of viral genes into any desired location in the viral genome.
It is a further object of this invention to provide a simple and useful system by which adenovirus cloning vectors may be developed.
It is a further object of this invention to provide a kit for efficient production of adenoviral vectors for vaccine and gene-therapeutic applications which relies on homologous or site-specific recombination, and which does not require a cold-chain for distribution.
A further object of this invention is to provide a system whereby the high-efficiency of the head-to-head ITR mediated recombination, with or without Cre-lox mediated recombination, is combined with enhanced infectivity achieved when adenovirus-TP complexes are utilized.
Further objects of this invention will become apparent from a review of the complete disclosure and the claims appended hereto.